Interception of host angiogenic signalling limits mycobacterial growth.

Pathogenic mycobacteria induce the formation of complex cellular aggregates called granulomas that are the hallmark of tuberculosis. Here we examine the development and consequences of vascularization of the tuberculous granuloma in the zebrafish-Mycobacterium marinum infection model, which is characterized by organized granulomas with necrotic cores that bear striking resemblance to those of human tuberculosis. Using intravital microscopy in the transparent larval zebrafish, we show that granuloma formation is intimately associated with angiogenesis. The initiation of angiogenesis in turn coincides with the generation of local hypoxia and transcriptional induction of the canonical pro-angiogenic molecule Vegfaa. Pharmacological inhibition of the Vegf pathway suppresses granuloma-associated angiogenesis, reduces infection burden and limits dissemination. Moreover, anti-angiogenic therapies synergize with the first-line anti-tubercular antibiotic rifampicin, as well as with the antibiotic metronidazole, which targets hypoxic bacterial populations. Our data indicate that mycobacteria induce granuloma-associated angiogenesis, which promotes mycobacterial growth and increases spread of infection to new tissue sites. We propose the use of anti-angiogenic agents, now being used in cancer regimens, as a host-targeting tuberculosis therapy, particularly in extensively drug-resistant disease for which current antibiotic regimens are largely ineffective.

Notes on recent experiments relating to the growth and rearing of food-fish at the laboratory

The rearing of lobster larvae and larval fish.

Male morph predicts investment in larval immune function in the dung beetle, Onthophagus taurus

Investment in immunity is costly, so that resource-based trade-offs between immunity and sexually selected ornaments might be expected. The amount of resources that an individual can invest in each trait will be limited by the total resources available to them. It would therefore be informative to investigate how investment in immune function changes during growth or production of the sexual trait as resources are diverted to it. Using the dung beetle, Onthophagus taurus, which displays both sexual and male dimorphism in horn size, we examined changes in one measure of immune function, phenoloxidase (PO) activity, in the hemolymph of larvae prior to and during horn growth. We found that PO levels differed between small- and large-horned males throughout the final instar prior to the point where investment in horn growth was taking place. PO levels in females were intermediate to the 2 male morphs. These differences could not be accounted for by differences in condition, measured as hemolymph protein levels and weight. We suggest that the observed differences might be associated with sex- and morph-specific variation in juvenile hormone levels.

Calmodulin disruption impacts growth and motility in juvenile liver fluke

Background: Deficiencies in effective flukicide options and growing issues with drug resistance make current strategies for liver fluke control unsustainable, thereby promoting the need to identify and validate new control targets in Fasciola spp. parasites. Calmodulins (CaMs) are small calcium-sensing proteins with ubiquitous expression in all eukaryotic organisms and generally use fluctuations in intracellular calcium levels to modulate cell signalling events. CaMs are essential for fundamental processes including the phosphorylation of protein kinases, gene transcription, calcium transport and smooth muscle contraction. In the blood fluke Schistosoma mansoni, calmodulins have been implicated in egg hatching, miracidial transformation and larval development. Previously, CaMs have been identified amongst liver fluke excretory-secretory products and three CaM-like proteins have been characterised biochemically from adult Fasciola hepatica, although their functions remain unknown.

Methods: In this study, we set out to investigate the biological function and control target potential of F. hepatica CaMs (FhCaMs) using RNAi methodology alongside novel in vitro bioassays.

Results: Our results reveal that: (i) FhCaMs are widely expressed in parenchymal cells throughout the forebody region of juvenile fluke; (ii) significant transcriptional knockdown of FhCaM1-3 was inducible by exposure to either long (~200 nt) double stranded (ds) RNAs or 27 nt short interfering (si) RNAs, although siRNAs were less effective than long dsRNAs; (iii) transient long dsRNA exposure-induced RNA interference (RNAi) of FhCaMs triggered transcript knockdown that persisted for ≥ 21 days, and led to detectable suppression of FhCaM proteins; (iv) FhCaM RNAi significantly reduced the growth of juvenile flukes maintained in vitro; (v) FhCaM RNAi juveniles also displayed hyperactivity encompassing significantly increased migration; (vi) both the reduced growth and increased motility phenotypes were recapitulated in juvenile fluke using the CaM inhibitor trifluoperazine hydrochloride, supporting phenotype specificity.

Conclusions: These data indicate that the Ca(2+)-modulating functions of FhCaMs are important for juvenile fluke growth and movement and provide the first functional genomics-based example of a growth-defect resulting from gene silencing in liver fluke. Whilst the phenotypic impacts of FhCaM silencing on fluke behaviour do not strongly support their candidature as new flukicide targets, the growth impacts encourage further consideration, especially in light of the speed of juvenile fluke growth in vivo.

Effects of environmental conditions on planktonic abundances, benthic recruitment and growth rates of the bivalve mollusc Ruditapes decussatus in a Portuguese coastal lagoon

Controlling environmental parameters for the early stages of marine invertebrates have received little attention, particularly in field studies. This study involves measurement of abundances and growth rates of the bivalve Ruditapes decussatus Linnaeus 1758 during its planktonic larval and early benthic life stages in a coastal lagoon: Ria Formosa, Portugal. Measured abundances were compared with tidal amplitude, water temperature, salinity, wind velocity and direction, and a food availability indicator 2chlorophyll a). Data were obtained on abundance and prodissoconch length of the larvae, measured two tothreetimes perweek,and ofpost-larval stagesindividuals thathadsettled inartificial collectors over10months.Larval and juvenile cohorts were identified using size-frequency distributions and larval ages estimated by larval shell growth lines.

A marine bacterium, Micrococcus MCCB 104, antagonistic to vibrios in prawn larval rearing systems

marine bacterium, Micrococcus MCCB 104, isolated from hatchery water, demonstrated extracellular antagonistic properties against Vibrio alginolyticus, V. parahaemolyticus, V. vulnificus, V. fluviallis, V. nereis, V. proteolyticus, V. mediterranei, V. cholerae and Aeromonas sp., bacteria associated with Macrobrachium rosenbergii larval rearing systems. The isolate inhibited the growth of V. alginolyticus during co-culture. The antagonistic component of the extracellular product was heat-stable and insensitive to proteases, lipase, catalase and α-amylase. Micrococcus MCCB 104 was demonstrated to be non-pathogenic to M. rosenbergii larvae

Development of nitrifying bacterial consortia for immobilizing in nitrifying bioreactors designed for penaeid and non-penaeid larval rearing systems in the tropics

Two ammonia oxidizing (AMOPCU-1 and AMONPCU-1) and two nitrite oxidizing (NIOPCU-1 and NIONPCU-1) consortia for activating nitrifying bioreactors and thereby establishing nitrification in penaeid and non-penaeid hatchery systems were developed by enrichment. For further amplification of the consortia a simple medium having seawater (either salinity 30 ‰ or 15 ‰) as base, supplemented with NH4+-N/NO2--N and PO4- and pH adjusted to 8 was identified. During the amplification in a fermentor the consortia exhibited excessive wall growth and diminished their yield coefficient posing difficulty in harvesting the cells completely. The consortia consisted of both Gram negative and Gram-positive bacterial cells embedded in a mucilaginous matrix of glycocalyx - like material presumably composed of polysaccharides. The consortia besides being useful in activating nitrifying bioreactors developed for shrimp/prawn hatchery systems can also be used as bioaugmentors in the bioremediation of ammonia and nitrite toxicity in aquaculture systems.

Disruption of Chrysomya megacephala Growth Caused by Lignan Grandisin

The toxicity of tetrahydrofuran lignan grandisin was evaluated against larvae of Chrysomya megacephala F. (Diptera: Calliphoridae). The bioassay involved topical treatment on larvae, topical treatment oil egg masses, and incorporation in the larval diet. Grandisin showed inhibition of postembryonic development by ovicidal (30%) and larvicidal (38%) effects and reduced larval weight (4 mg), when topically applied oil egg masses and starving larvae (L1) at a concentration of 100 mu g/mu l. These findings elucidated the effect of grandisin on the C. megacephala life cycle and its potential to control C. megacephala populations.

Larval development of himri, Barbus luteus (Cyprinidae: Cypriniformes) reared in the laboratory

Larval development of the himri barbel, Barbus luteus Heckel, reared at 19-22 ¡C in natural daylight is described. Morphological and some functional character appearances were similar to the main ontogenetic steps of development in most cyprinids. Small hatched larvae attained both metamorphosis and a length of 10.5 mm after almost 35 days. Relative growth of the selected body proportions demonstrated typical priorities in growth compared to the length. An ontogenetic index was inferred from applying age and length criteria at metamorphosis as a scale for ontogenetic events.

Changes in the nutritional composition of captive early-mid stage Panulirus ornatus phyllosoma over ecdysis and larval development

Elucidation of the key nutritional requirements for complete larval development of the tropical spiny rock lobster, Panulirus ornatus, presents a major challenge for the development of robust commercial aquaculture for this crustacean. As a foundation study in this area, the chemical composition of early-mid stage P. ornatus phyllosoma (Stages I-VI) receiving a novel formulated diet was analysed immediately prior and post-ecdysis to provide insight into the crude nutritional trends during the larval development cycle. From the onset of moulting, cyclical patterns were evident in the proximate composition of phyllosoma, resulting in substantial restructuring between the pre- and post-moult stages of the moult cycle. Proportions of protein, lipid and ash were high at the premoult stage, reflecting growth and nutrient accumulation over the intermoult period, and reduced at the post-moult stage, reflecting the large uptake of water to facilitate subsequent growth. Polar lipid was the dominant lipid class, accounting for >. 90% of the total lipid content. Conversely, triacylglycerol concentrations were low (<. 5%), despite being the principal lipid class available in the formulated diet. Likewise, despite receiving high concentrations of eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) (9.2 and 7.6% of the dietary lipid source, respectively), levels of these fatty acids were comparatively low in phyllosoma (3.4 and 4.7%, respectively). In contrast, there is selective deposition of these fatty acids in wild caught phyllosoma. This finding suggests a poor assimilation of triacylglycerols by captive larvae and highlights the importance of future investigations into alternative sources of EPA and DHA. Ultimately, this study provides insight into the nutritional requirements of phyllosoma, providing valuable knowledge on diet formulation for commercially viable hatchery production of spiny rock lobsters. © 2014 Elsevier B.V.

Influence of food restriction on the reproduction and larval performance of matrinxã, Brycon amazonicus (Spix and Agassiz, 1829)

Este estudo avaliou o efeito da restrição alimentar e realimentação na reprodução de fêmeas e no crescimento inicial e sobrevivência de larvas de matrinxã, Brycon amazonicus. Matrizes distribuídas em 8 viveiros (15 peixes/tanque) foram alimentadas diariamente (em 4 tanques - G1) e alimentados em ciclos de 3 dias de alimentação seguidos de 2 dias de restrição (em 4 tanques - G2) por 6 meses antes da desova. Na indução à desova, 57% das fêmeas no G1 e 45% no G2 desovaram. Os pesos médios dos oócitos foram 208,1 g (G1) e 131,6 g (G2), sendo os oócitos G2 menores (1,017 ± 0,003 mm) que os oócitos de G1 (1,048 ± 0,002 mm). As taxas de fertilização (71,9 ± 12,6% e 61,2 ± 13,7%) e de eclosão (61,3 ± 33,9% e 67,5 ± 23,4%) entre os G1 e G2 não diferiram. Larvas foram coletadas na eclosão e às 24, 48 e 72 horas de incubação para medida do crescimento e as restantes transferidas para aquários e amostradas 1, 5, 9 e 15 dias depois. Na transferência, as larvas G1 e G2 tinham pesos similares (1,5 ± 0,15 e 1,46 ± 0,07 mg), mas o comprimento das larvas G2 era maior (6,2 ± 0,13 e 6,7 ± 0,14 mm). Ao 9° dia, quando é recomendada a transferência dos juvenis para tanques externos, os juvenis G2 tinham peso (13,6 ± 0,26 e 18,9 ± 0,07 mg) e comprimento (11,8 ± 0,09 e 14,5 ± 0,04 mm) maiores, mas no 15º dia os juvenis G1 eram maiores em peso (90,2 ± 1,19 e 68,6 ± 0,77 mg) e comprimento (18,8 ± 0,16 e 18,5 ± 0,04 mm). Aos 15 dias, a prole das fêmeas submetidas à restrição alimentar apresentou sobrevivência mais alta que a prole das fêmeas alimentadas diariamente (24,7 ± 2,07% e 19,2 ± 1,91%). A restrição alimentar imposta às fêmeas de matrinxã, apesar de reduzir o número de fêmeas que desovaram e a quantidade de oócitos extrusados, não afetou a fertilização e eclosão das larvas e melhorou a sobrevivência final das larvas.

Morphological and histochemical study of the myotomal muscle in pacu (Piaractus mesopotamicus Holmberg, 1887) during the initial growth phases

Morphological and histochemical methods were used to evaluate the myotomal muscle characteristics of pacu (Piaractus mesopotamicus) from hatching to 40 days old. During the larval period, the musculature consisted predominantly of white muscle. White and red muscle mass increased at 10, 20, 30 and 40 days after hatching. The larvae had round muscle fibers with a moderate degree of maturation and central nuclei. In subsequent phases, small and immature fibers were visible near larger and more differentiated fibers. Undifferentiated cells or presumptive myoblasts located in embryogenic zones were visible in the dorsal and ventral regions, and were more evident at 30 and 40 days. The red muscle fibers located in the subdermal region, had oxidative metabolism and slow contraction, whereas the more predominant white muscle fibers had glycolytic metabolism and fast contraction. Our findings indicate that during the initial phases, myotomal muscle growth in pacu occurs by both, muscle fiber hypertrophy and hyperplasia. The analysis of frequency of red and white muscle fibers shows that hyperplastic growth is intense in this period. As the growth rate in adult fish is related to the number of muscle fibers in young fish, extrinsic factors could change the muscle fiber phenotype and influence their ultimate size.

The influence of initial feeding on muscle development and growth in pacu Piaractus mesopotamicus larvae

The effects of different feeding schemes on pacu Piaractus mesopotamicus early development were evaluated with respect to growth, survival, muscle development, and differential gene expression of MyoD and myogenin. The pacu larvae (4 days post hatch-dph, 0.77 mg wet weight) were given six feeding treatments intentionally designed to cause variations in the larvae growth rate: (A) only artemia nauplii; (CD) only a commercial diet; (ED) only a semi-purified experimental diet; (ACD) and (AED) two treatments that involved weaning; and (S) starvation. Early weaning from artemia nauplii to the formulated diets (ACD and AED) affected growth and survival of the pacu larvae compared with the exclusive use of artemia (A). Starvation (S) and the commercial diet (CD) caused total mortality in pacu larvae at 18 dph. The experimental diet (ED) assured low fish survival and growth. The skeletal muscle morphology was not affected by the delay in somatic growth from early weaning onto the formulated diets. Three distinct muscle compartments were observed throughout the larval development in treatments A, ACD and AED: superficial, deep and intermediate, accompanied by muscle thickening. Severe undernourishment caused drastic differences in growth and in the morphology of the muscle fibers. Pacu larvae fed only formulated diets (CD and ED) showed muscle characteristics similar to the larvae in starvation (S) during the first 15 dph. At 27 and 35 dph, a slight increase in epaxial muscle mass was noted in larvae fed only the experimental diet (ED). At 35 dph, we observed a high frequency of fibers >= 40 mu m in the larvae that were weaned onto the formulated diets (ACD and AED), indicative of hypertrophy. In contrast, the larvae fed only artemia nauplii (A) displayed a larger number of fibers with diameters <= 20 mu m, which is indicative of hyperplasia. The expression of the MyoD and myogenin genes in pacu larvae at 35 dph was not affected by initial feeding (p>0.05). In conclusion, the formulated diets used impaired pacu larvae growth and survival; therefore, they were inadequate for pacu, at least at the times they were introduced. Artemia nauplii were the most adequate food source during first feeding of the pacu, and they produced bigger fish upon completion of the experiment. Moreover, the contribution of hyperplasia to the skeletal muscle growth appeared higher in fast- than in slow-growing pacu larvae. (C) 2011 Elsevier By. All rights reserved.

Postlarval stages and growth patterns of the spider crab Pyromaia tuberculata (Brachyura, Majidae) from laboratory-reared material

The spider crab Pyromaia tuberculata was introduced into southeastern Brazil; ovigerous material was collected and reared in the laboratory. Morphologic changes and growth patterns of post-larval development are reported. Results show that within-stage size variation is lowest in mature stages, especially in the case of females in which there is an apparent size threshold for the last juvenile stages to undergo the puberty molt. A prepuberty molt taking place at the fourth crab stage is indicated by analyzing the allometric growth of the abdomen in females. In contrast, the same procedure using the allometric growth of chelae failed in detecting both the prepuberty and puberty molts in males. Conversely to females, which develop a complex brood chamber at the puberty molt, the enlargement of chelae was not consistent in all postpuberty males. The short instar sequence of this species, in no case exceeding nine stages, is marked by conspicuous morphologic alterations achieved at each molt. Almost all stages can be identified by examining diagnostic features of rostrum, abdomen, sternum, and pleopods.

Juvenile development and growth patterns in the mud crab Eurytium limosum (Say, 1818) (Decapoda, Brachyura, Xanthidae) under laboratory conditions

The post-larval development of the mud crab Eurytium limosum was studied under laboratory conditions by using the offspring of ovigerous females collected at the Comprido River mangrove, SP, Brazil. The first crab stage is fully described and the juvenile development, until crab stage 10, is examined with emphasis on morphological change, sexual differentiation and growth patterns. The carapace of the first crab stage is nearly square as observed in other xanthids, becoming similar to adults only at stage 15. The sexes can be distinguished from stage four, based on the number of pleopods and their morphology. While the intermoult period increases, the moult percentage decreases at each stage. The abdominal allometric growth is sex-dependent, with males showing a negative (b=0.71) and females an isometric (b=0.95) relative growth pattern. Male gonopods undergo a positive allometric growth, and their shape changes remarkably until sexual maturity. The cheliped dentition can be observed after stage 4. Regardless of sex, most crabs have a molariform right cheliped, which is thought to aid the handling of asymmetric prey such as gastropods.